Aptamers are short oligonucleotides (single stranded DNA or RNA) which can form three-dimensional structures that specifically bind with high affinity and specificity to a wide range of targets including, for example, proteins, organic molecules, and inorganic molecules (Ellington and Szostak, 1990; Tuerk and Gold, 1990). The binding affinities of aptamers to proteins are similar or even higher than those of antibodies with typical dissociation constants (Kd values) of micromolar to low picomolar range (Berezovski et al., 2005; Drabovich et al., 2005; Jensen et al., 1995; Mendonsa et al., 2005, 2005; Morris et al., 1998; Tuerk and Gold, 1990). Some aptamers exhibit femtomolar affinity.
Compared to antibodies, aptamers are easier to produce and inexpensive since the generation process occurs in vitro without the need for animals. In theory, aptamers can be generated against any target protein, and the binding target site of the protein can be determined. Once sequenced, aptamers can be synthesized at lower cost than antibodies, and may display lower cross-reactivity for a given target than an antibody. Aptamers can be easily modified with different chemical groups to enhance chemical properties such as stability or resolvability, and to achieve various functions. Aptamer coated surfaces can often be heated and reused.
The conventional approach for generating aptamers is through systematic evolution of ligands by exponential enrichment (SELEX) by which target-specific aptamers are selected and synthesized in vitro from a random aptamer library (Ellington and Szostak, 1990; Tuerk and Gold, 1990). SELEX typically involves incubation of ligand sequences with a target; partitioning of ligand-target complexes from unbound sequences via affinity methods; and amplification of bound sequences. In the incubation step, nucleic acid libraries are incubated with target molecules in an appropriate buffer at a desired temperature. After binding, the RNA/ssDNA aptamer-target complexes are separated from nonspecific molecules. Bound sequences are regenerated by enzymatic amplification processes. The amplified molecules are then used in the next round of selection. Selecting sequences which have the highest specificity and affinity against the target typically requires eight to twelve cycles. The selected oligonucleotides are analyzed for their sequences and structures after cloning and sequencing. After the sequence of an aptamer is determined, the aptamer can be easily generated through nucleic acid synthesis, and its binding affinity and specificity to a specific target can be validated. Aptamers may then be used in a variety of analytical, bioanalytical, therapeutic and diagnostic applications including, for example, protein identification and purification; inhibition of receptors or enzyme activities; and detection of proteins from bacteria in environmental or clinical samples.
Group A streptococcus (GAS) is implicated in a variety of ailments, including streptococcal pharyngitis, necrotizing fascitis, scarlet fever, streptococcal toxic shock syndrome, invasive systemic infections, and endocarditis (CDC, 2007). Usually throat and skin swabs, and wound aspirate from patients are tested. Point-of-care testing methodology relies upon either culture or antibody-based Rapid Antigen Detection (RAD), particularly a two-site sandwich immunoassay, to detect the Group A cell wall carbohydrate. Culture requires at minimum six to eight hours overnight incubation (Leung et al., 2006). In comparison, RAD is quicker, taking only minutes but having poor sensitivity and requiring a confirmatory culture step following negative results (Armengol et al., 2004). The sensitivity of culture and RAD is dependent upon the presence of a sufficient number of live cells in the inoculums.
An aptamer-based RAD test could negate the need for a confirmatory culture step, and may also provide lower cross-reactivity than antibody-based methods. In addition to replacing antibodies in RAD, aptamers against GAS cell surface molecules could prove useful in other assay formats. Current assay formats for bacterial detection using aptamers include enzyme-linked oligonucleotide assays, flow cytometry, chemiluminescent sandwich aptasensors, aptamer-quantum dot conjugates, and FRET-based assays (Bruno et al., 1999, 2002, 2010; Chen et al., 2007; Ikanovic et al., 2007; Fan et al., 2008; Hamula et al., 2008). The flexibility of aptamer reagents may enable the development of efficient, sensitive point-of-care diagnostic assays for GAS; for example, epidemiological surveillance of GAS clinical isolates is important for outbreak management, and vaccine development and implementation.
One of the major virulence factors of invasive GAS isolates (iGAS) is the M protein which is present on the bacterial surface (Beachey et al., 1981; Fischetti, 1991; Jones and Fischetti, 1988; Lancefield, 1962). While the M protein is a critical virulence factor for GAS, it can also be utilized as a typing marker for understanding the epidemiology of iGAS disease. The M protein can be typed serologically. Alternatively, GAS can be typed by sequencing of the gene which encodes the M protein, the emm gene (Beall et al., 2000; Whatmore et al., 1994). Currently, emm nomenclature extends from emm1 to emm124, with many emm types having minor variations in the nucleotide coding sequence resulting in emm subtypes for a particular emm type (Beall et al., 1996, 2000; Facklam et al., 1999; Neal et al., 2007). Different M-types are often, but not always, associated with different invasive infections; for example, M1 and M3 are more often associated with invasive infections than other M-types (Sharkawy et al., 2002; Vlaminckx et al., 2003).
Conventionally, GAS is M-typed via precipitin or latex agglutination methods, which involve screening bacterial surface extracts against different M-protein specific reference polyclonal antisera or antibodies conjugated to latex beads (Lancefield, 1933). Consistency between batches of typing sera is low (Facklam and Moody, 1968). Sequencing of the emm gene is replacing antibody-based typing methods and has expanded the repertoire of GAS emm types worldwide (Beall et al., 1996; Gardiner et al., 1995; Kaufhold et al., 1994; Saunders et al., 1997). Due to the complexity of such methods, the M typing of GAS isolates is conducted in laboratories specializing in GAS characterization. However, these methods are laborious and have low-throughput since each requires comparison of a bacterial isolate to a myriad of reference strains or databases.
The protein-based serotyping system of GAS makes it an ideal aptamer target. The M-protein contains a hypervariable N-terminus, to which typing antibodies bind to distinguish one M-type from another (Fischetti, 1989). Proteins are more successful SELEX targets than other smaller types of cell surface molecules such as carbohydrates and lipids. A SELEX technique has been developed against whole, live bacterial cells (Stoltenburg et al., 2007). Aptamers which bind to bacterial cell surface molecules using live L. acidophilus have been described (Hamula et al., 2008). However, the starting library diversity contained only 1012 to 1013 different sequences and required heat denaturation or streptavidin-biotin mediated separation to render the library and aptamer pools single-stranded.